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1.  Evaluation of candidate spermatogonial markers ID4 and GPR125 in testes of adult human cadaveric organ donors 
Andrology  2014;2(4):607-614.
The optimal markers for human spermatogonial stem cells (SSCs) are not known. Among the genes recently linked to SSCs in mice and other animals are the basic helix-loop-helix transcription factor ID4 and the orphan G-protein coupled receptor GPR125. While ID4 and GPR125 are considered putative markers for SSCs, they have not been evaluated for co-expression in human tissue. Further, neither the size nor the character of the human spermatogonial populations that express ID4 and GPR125, respectively, are known. A major barrier to addressing these questions is the availability of healthy adult testis tissue from donors with no known reproductive health problems. To overcome this obstacle, we have employed healthy testicular tissue from a novel set of organ donors (n=16; aged 17-68 years) who were undergoing post-mortem clinical organ procurement. Using immunolabeling, we found that ID4 and GPR125 are expressed on partially overlapping spermatogonial populations and are more broadly expressed in the normal adult human testis. Additionally, we found that expression of ID4 remained stable during aging. These findings suggest that ID4 and GPR125 could be efficacious for identifying previously unrecognized human spermatogonial subpopulations in conjunction with other putative human stem cell markers, both in younger and older donors.
PMCID: PMC4153397  PMID: 24902969
2.  Transmission of Multidrug-Resistant and Drug-Susceptible Tuberculosis within Households: A Prospective Cohort Study 
PLoS Medicine  2015;12(6):e1001843.
The “fitness” of an infectious pathogen is defined as the ability of the pathogen to survive, reproduce, be transmitted, and cause disease. The fitness of multidrug-resistant tuberculosis (MDRTB) relative to drug-susceptible tuberculosis is cited as one of the most important determinants of MDRTB spread and epidemic size. To estimate the relative fitness of drug-resistant tuberculosis cases, we compared the incidence of tuberculosis disease among the household contacts of MDRTB index patients to that among the contacts of drug-susceptible index patients.
Methods and Findings
This 3-y (2010–2013) prospective cohort household follow-up study in South Lima and Callao, Peru, measured the incidence of tuberculosis disease among 1,055 household contacts of 213 MDRTB index cases and 2,362 household contacts of 487 drug-susceptible index cases.
A total of 35/1,055 (3.3%) household contacts of 213 MDRTB index cases developed tuberculosis disease, while 114/2,362 (4.8%) household contacts of 487 drug-susceptible index patients developed tuberculosis disease. The total follow-up time for drug-susceptible tuberculosis contacts was 2,620 person-years, while the total follow-up time for MDRTB contacts was 1,425 person-years. Using multivariate Cox regression to adjust for confounding variables including contact HIV status, contact age, socio-economic status, and index case sputum smear grade, the hazard ratio for tuberculosis disease among MDRTB household contacts was found to be half that for drug-susceptible contacts (hazard ratio 0.56, 95% CI 0.34–0.90, p = 0.017). The inference of transmission in this study was limited by the lack of genotyping data for household contacts. Capturing incident disease only among household contacts may also limit the extrapolation of these findings to the community setting.
The low relative fitness of MDRTB estimated by this study improves the chances of controlling drug-resistant tuberculosis. However, fitter multidrug-resistant strains that emerge over time may make this increasingly difficult.
In this prospective cohort study, Louis Grandjean and colleagues examine the relative fitness of multidrug-resistant versus drug-susceptible tuberculosis for transmission among household contacts in South Lima and Callao, Peru.
Editors' Summary
Tuberculosis—a contagious bacterial disease that usually infects the lungs—is a global public health problem. Every year, 8.6 million people develop active tuberculosis (tuberculosis disease), and at least 1.3 million people die as a result, mainly in resource-limited countries. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is spread in airborne droplets when people with tuberculosis disease cough or sneeze. Consequently, an individual’s risk of contracting tuberculosis increases with his/her frequency of contact with people who have the disease; people who live in the same household as someone with tuberculosis disease are at particularly high risk. Other risk factors for contracting tuberculosis include living in crowded or insanitary conditions and being immunocompromised because of, for example, infection with HIV. The characteristic symptoms of tuberculosis disease are persistent cough, fever, weight loss, and night sweats. Diagnostic tests for the disease include sputum smear microscopy (microscopic analysis of mucus coughed up from the lungs), the growth of M. tuberculosis from sputum samples, and chest X-rays.
Why Was This Study Done?
Taking several antibiotics (including rifampicin and isoniazid) daily for six months can cure tuberculosis, but the emergence of multidrug-resistant tuberculosis (MDRTB) is making the disease increasingly hard to treat. How badly MDRTB will affect tuberculosis control efforts is likely to depend on the relative “fitness” of multi-drug resistant and drug-susceptible M. tuberculosis strains. The fitness of a pathogen (infectious organism) is its ability to survive, reproduce, be transmitted, and cause disease in another host. Animal and laboratory studies indicate that drug-resistant M. tuberculosis strains are less fit than drug-susceptible strains, but these studies do not account for the clinical, environmental, and socio-economic variables that influence a patient’s ability to cause tuberculosis disease in a contact, and may not accurately measure the relative fitness of M. tuberculosis strains. In this prospective cohort study, the researchers estimate the fitness of drug-resistant tuberculosis relative to drug-susceptible tuberculosis by comparing the incidence of additional cases of tuberculosis disease in households with an MDRTB index case and the incidence in households with a drug-susceptible tuberculosis index case. A prospective cohort study follows a group of people over time to see whether specific baseline characteristics are associated with specific outcomes. The incidence of a disease is the number of new cases in a population over a given time period.
What Did the Researchers Do and Find?
The researchers enrolled 1,055 household contacts of 213 MDRTB index cases (individuals whose disease was resistant to at least rifampicin and isoniazid) and 2,362 household contacts of 487 drug-susceptible tuberculosis index cases living in South Lima and Callao, Peru. During three years of follow-up, 35 (3.3%) of the household contacts of the MDRTB index cases and 114 (4.8%) of the household contacts of the drug-susceptible tuberculosis index cases developed tuberculosis disease. After adjusting for factors likely to affect the transmission of tuberculosis, such as HIV status, socio-economic status, and sputum smear grade of the index case (higher smear grades are associated with a higher risk of tuberculosis transmission), the hazard ratio for tuberculosis disease for household contacts of MDRTB index cases was half that of the household contacts of drug-susceptible tuberculosis index cases. That is, the household contacts of MDRTB index cases contracted tuberculosis disease half as often as those of drug-susceptible tuberculosis index cases.
What Do These Findings Mean?
These findings indicate that, within households, MDRTB has a relatively low fitness compared to drug-susceptible tuberculosis. That is, at least during the first three years following exposure, individuals with MDRTB are less likely to transmit disease to their household contacts than individuals with drug-susceptible tuberculosis. These findings agree with those of previous animal and laboratory studies and with the findings of molecular epidemiology studies that have used genetic methods to estimate M. tuberculosis fitness within populations. Because the researchers did not genetically compare M. tuberculosis strains isolated from the index cases with strains isolated from the household contacts who developed tuberculosis disease, some of these contacts may have become infected outside the household. Moreover, it may not be possible to extrapolate these findings to the community setting. Nevertheless, the low relative fitness of MDRTB reported here improves our chances of controlling the spread of drug-resistant tuberculosis, with the proviso that the emergence of fitter MDRTB strains over time might yet threaten global tuberculosis control efforts.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at
The World Health Organization provides information (in several languages) on tuberculosis and on multidrug-resistant tuberculosis; the Global Tuberculosis Report 2014 provides information about tuberculosis around the world; a supplement to the report entitled Drug-Resistant TB—Surveillance and Response is available
The Stop TB Partnership is working towards tuberculosis elimination and provides personal stories about tuberculosis (in English and Spanish); the Tuberculosis Vaccine Initiative (a not-for-profit organization) also provides personal stories about tuberculosis
The US Centers for Disease Control and Prevention provides information about tuberculosis and about drug-resistant tuberculosis (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
PMCID: PMC4477882  PMID: 26103620
3.  High-dose rifampicin kills persisters, shortens treatment duration, and reduces relapse rate in vitro and in vivo 
Although high-dose rifampicin holds promise for improving tuberculosis control by potentially shortening treatment duration, these effects attributed to eradication of persistent bacteria are unclear. The presence of persistent Mycobacterium tuberculosis was examined using resuscitation promoting factors (RPFs) in both in vitro hypoxia and in vivo murine tuberculosis models before and after treatment with incremental doses of rifampicin. Pharmacokinetic parameters and dose-dependent profile of rifampicin in the murine model were determined. The Cornell mouse model was used to test efficacy of high-dose rifampicin in combination with isoniazid and pyrazinamide and to measure relapse rate. There were large numbers of RPF-dependent persisters in vitro and in vivo. Stationary phase cultures were tolerant to rifampicin while higher concentrations of rifampicin eradicated plate count positive but not RPF-dependent persistent bacteria. In murine infection model, incremental doses of rifampicin exhibited a dose-dependent eradication of RPF-dependent persisters. Increasing the dose of rifampicin significantly reduced the risk of antibiotic resistance emergence. In Cornell model, mice treated with high-dose rifampicin regimen resulted in faster visceral clearance; organs were M. tuberculosis free 8 weeks post-treatment compared to 14 weeks with standard-dose rifampicin regimen. Organ sterility, plate count and RPF-dependent persister negative, was achieved. There was no disease relapse compared to the standard dose regimen (87.5%). High-dose rifampicin therapy results in eradication of RPF-dependent persisters, allowing shorter treatment duration without disease relapse. Optimizing rifampicin to its maximal efficacy with acceptable side-effect profiles will provide valuable information in human studies and can potentially improve current tuberculosis chemotherapy.
PMCID: PMC4477163  PMID: 26157437
Mycobacterium tuberculosis; rifampicin; persistence; resuscitation promoting factors; mouse model
4.  Understanding paratyphoid infection: study protocol for the development of a human model of Salmonella enterica serovar Paratyphi A challenge in healthy adult volunteers 
BMJ Open  2015;5(6):e007481.
This study will develop the first human challenge model of paratyphoid infection which may then be taken forward to evaluate paratyphoid vaccine candidates. Salmonella Paratyphi A is believed to cause a quarter of the estimated 20 million cases of enteric fever annually. Epidemiological evidence also suggests that an increasing proportion of the enteric fever burden is attributable to S. Paratyphi infection meriting further attention and interest in vaccine development. Assessment of paratyphoid vaccine efficacy in preclinical studies is complicated by the lack of a small animal model and the human-restricted nature of the infection. The use of experimental human infection in healthy volunteers provides an opportunity to address these problems in a cost-effective manner.
Methods and analysis
Volunteers will ingest virulent S. Paratyphi A bacteria (NVGH308 strain) with a bicarbonate buffer solution to establish the infectious dose resulting in an ‘attack rate’ of 60–75%. Using an a priori decision-making algorithm, the challenge dose will be escalated or de-escalated to achieve the target attack rate, with the aim of reaching the study end point while exposing as few individuals as possible to infection. The attack rate will be determined by the proportion of paratyphoid infection in groups of 20 healthy adult volunteers, with infection being defined by one or more positive blood cultures (microbiological end point) and/or fever, defined as an oral temperature exceeding 38°C sustained for at least 12 h (clinical end point); 20–80 participants will be required. Challenge participants will start a 2-week course of an oral antibiotic on diagnosis of infection, or after 14 days follow-up.
Ethics and dissemination
The strict eligibility criterion aims to minimise risk to participants and their close contacts. Ethical approval has been obtained. The results will be disseminated in a peer-reviewed journal and presented at international congresses.
Trial registration number
PMCID: PMC4480031  PMID: 26082464
5.  Can Neural Activation in Dorsolateral Prefrontal Cortex Predict Responsiveness to Information? An Application to Egg Production Systems and Campaign Advertising 
PLoS ONE  2015;10(5):e0125243.
Consumers prefer to pay low prices and increase animal welfare; however consumers are typically forced to make tradeoffs between price and animal welfare. Campaign advertising (i.e., advertising used during the 2008 vote on Proposition 2 in California) may affect how consumers make tradeoffs between price and animal welfare. Neuroimaging data was used to determine the effects of brain activation in dorsolateral prefrontal cortex (dlPFC) on choices making a tradeoff between price and animal welfare and responsiveness to campaign advertising. Results indicated that activation in the dlPFC was greater when making choices that forced a tradeoff between price and animal welfare, compared to choices that varied only by price or animal welfare. Furthermore, greater activation differences in right dlPFC between choices that forced a tradeoff and choices that did not, indicated greater responsiveness to campaign advertising.
PMCID: PMC4446318  PMID: 26018592
6.  The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru 
PLoS ONE  2015;10(5):e0126271.
The comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.
To investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.
The Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR's 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).
Tuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.
PMCID: PMC4435908  PMID: 25984723
7.  Neuroimaging Studies of Factors Related to Exercise: Rationale and design of a 9 month trial 
Contemporary clinical trials  2013;37(1):58-68.
The prevalence of obesity is high resulting from chronic imbalances between energy intake and expenditure. On the expenditure side, regular exercise is associated with health benefits, including enhanced brain function. The benefits of exercise are not immediate and require persistence to be realized. Brain regions associated with health-related decisions, such as whether or not to exercise or controlling the impulse to engage in immediately rewarding activities (e.g., sedentary behavior), include reward processing and cognitive control regions. A 9 month aerobic exercise study will be conducted in 180 sedentary adults (n = 90 healthy weight [BMI= 18.5 to 26.0 kg/m2]; n = 90 obese [BMI=29.0 to 41.0 kg/m2) to examine the brain processes underlying reward processing and impulse control that may affect adherence in a new exercise regimen. The primary aim is to use functional magnetic resonance imaging (fMRI) to examine reward processing and impulse control among participants that adhere (exercise >80% of sessions) and those that do not adhere to a nine-month exercise intervention with secondary analyses comparing sedentary obese and sedentary healthy weight participants. Our results will provide valuable information characterizing brain activation underlying reward processing and impulse control in sedentary obese and healthy weight individuals. In addition, our results may identify brain activation predictors of adherence and success in the exercise program along with measuring the effects of exercise and improved fitness on brain activation.
PMCID: PMC3946871  PMID: 24291150
Exercise Adherence; Functional Magnetic Resonance Imaging; Obesity
8.  Tonic Hyper-Connectivity of Reward Neurocircuitry in Obese Children 
Obesity (Silver Spring, Md.)  2014;22(7):1590-1593.
Obese children demonstrate less activation in prefrontal regions associated with self-control and inhibition when presented with food cues and advertisements. The current study evaluates the differences between obese and healthy weight children in resting-state functional connectivity to these brain regions.
Design and Methods
Seed regions in bilateral middle frontal gyri were chosen based on previous task-based analysis showing differences between obese and healthy weight children’s responses to food-associated stimuli. Functional connectivity to these seed regions was measured in resting-state scans collected in obese and lean children undergoing fMRI.
Obese children exhibited greater resting-state functional connectivity than healthy weight children between the left middle frontal gyrus and reward-related regions in the left ventromedial prefrontal cortex, as well as the left lateral OFC.
Previously published results demonstrate that obese children exhibit less activity in brain regions associated with self-control when viewing motivationally salient food advertisements. Here we show that obese children also have tonically greater input to these self-control regions from reward neurocircuitry. The greater functional connectivity between reward and self-control regions, in conjunction with weaker activation of self-control neurocircuitry, may render these children more susceptible to food advertisements, placing them at elevated risk for over-feeding and obesity.
PMCID: PMC4077951  PMID: 24634397
children; functional magnetic resonance imaging (fMRI); resting-state; impulsivity; orbitofrontal cortex; ventromedial prefrontal cortex
9.  Evening Hyperphagia and Food Motivation: A Preliminary Study of Neural Mechanisms 
Eating behaviors  2013;14(4):10.1016/j.eatbeh.2013.08.006.
Evening hyperphagia (EH; consumption of ≥ 25% of total daily calories after the evening meal) is a circadian delay in the pattern of daily food intake and is a core criterion of night eating syndrome (Allison et al., 2010). This preliminary study examined the brain response to food cues using functional magnetic resonance imaging (fMRI) in seven obese adults with EH compared to seven obese adults without EH. When contrasting food to non-food and blurry baseline images pre-meal, groups differed in brain activation in the inferior frontal gyrus, precentral gyrus, cingulate gyrus, superior temporal gyrus and cerebellum. At post meal, groups differed in brain activation in the fusiform gyrus, inferior frontal gyrus, inferior parietal lobule and the cerebellum. Significant interactions between time (pre-meal, post-meal) and group (EH, control) when contrasting food to non-food images were also noted in the inferior frontal gyrus and the superior temporal gyrus. Further research is necessary to replicate these findings and determine if they have a mechanistic role in the development of circadian delayed eating behavior in obese adults with EH.
PMCID: PMC3817498  PMID: 24183133
evening hyperphagia; food motivation; disinhibition; inferior frontal gyrus; neural mechanisms; functional magnetic resonance imaging
10.  Winning and losing: differences in reward and punishment sensitivity between smokers and nonsmokers 
Brain and Behavior  2014;4(6):915-924.
Smokers show increased brain activation in reward processing regions in response to smoking-related cues, yet few studies have examined secondary rewards not associated with smoking (i.e., money). Inconsistencies exist in the studies that do examine secondary rewards with some studies showing increased brain activation in reward processing brain regions, while others show decreased activation or no difference in activation between smokers and nonsmokers.
The goal of the current study is to see if smokers process the evaluation and delivery of equally salient real world rewards similarly or differently than nonsmokers.
The current study employed functional magnetic resonance imaging (fMRI) to examine brain responses in smokers and nonsmokers during the evaluation and delivery of monetary gains and losses.
In comparison to nonsmokers, smokers showed increased activation in the ventromedial prefrontal cortex to the evaluation of anticipated monetary losses and the brain response. Moreover, smokers compared to nonsmokers showed decreased activation in the inferior frontal gyrus to the delivery of expected monetary gains. Brain activations to both the evaluation of anticipated monetary losses and the delivery of expected monetary gains correlated with increased self-reported smoking craving to relieve negative withdrawal symptoms and craving related to positive aspects of smoking, respectively.
Together these results indicate that smokers are hyperresponsive to the evaluation of anticipated punishment and hyporesponsive to the delivery of expected rewards. Although further research is needed, this hypersensitivity to punishments coupled with increased craving may negatively impact quit attempts as smokers anticipate the negative withdrawal symptoms associated with quitting.
PMCID: PMC4178298  PMID: 25365800
fMRI; punishment sensitivity; reward sensitivity; smoking
11.  Winning and losing: differences in reward and punishment sensitivity between smokers and nonsmokers 
Brain and Behavior  2014;10.1002/brb3.285.
Smokers show increased brain activation in reward processing regions in response to smoking‐related cues, yet few studies have examined secondary rewards not associated with smoking (i.e., money). Inconsistencies exist in the studies that do examine secondary rewards with some studies showing increased brain activation in reward processing brain regions, while others show decreased activation or no difference in activation between smokers and nonsmokers.
The goal of the current study is to see if smokers process the evaluation and delivery of equally salient real world rewards similarly or differently than nonsmokers.
The current study employed functional magnetic resonance imaging (fMRI) to examine brain responses in smokers and nonsmokers during the evaluation and delivery of monetary gains and losses.
In comparison to nonsmokers, smokers showed increased activation in the ventromedial prefrontal cortex to the evaluation of anticipated monetary losses and the brain response. Moreover, smokers compared to nonsmokers showed decreased activation in the inferior frontal gyrus to the delivery of expected monetary gains. Brain activations to both the evaluation of anticipated monetary losses and the delivery of expected monetary gains correlated with increased self‐reported smoking craving to relieve negative withdrawal symptoms and craving related to positive aspects of smoking, respectively.
Together these results indicate that smokers are hyperresponsive to the evaluation of anticipated punishment and hyporesponsive to the delivery of expected rewards. Although further research is needed, this hypersensitivity to punishments coupled with increased craving may negatively impact quit attempts as smokers anticipate the negative withdrawal symptoms associated with quitting.
PMCID: PMC4178298  PMID: 25365800
fMRI; punishment sensitivity; reward sensitivity; smoking
12.  A Field Evaluation of the Hardy TB MODS Kit™ for the Rapid Phenotypic Diagnosis of Tuberculosis and Multi-Drug Resistant Tuberculosis 
PLoS ONE  2014;9(9):e107258.
Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory.
Methods & Findings
2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3–99.8%), 98.3% (97.5–98.8%), 95.8% (94.0–97.1%), and 99.7% (99.3–99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9–13) days for conventional MODS and 8.5 (7–11) for MODS Kit (p<0.01). Direct rifampicin and isoniazid DST in MODS kit was almost universally concordant with conventional MODS (97.9% agreement, 665/679 evaluable samples) and reference indirect DST (97.9% agreement, 687/702 evaluable samples).
MODS kit delivers performance indistinguishable from conventional MODS and offers a convenient, affordable alternative with enhanced safety from the sealing silicone lid. The availability in the marketplace of this platform, which conforms to European standards (CE-marked), readily repurposed for second-line DST in the near future, provides a fresh opportunity for improving equity of access to TB diagnosis and first and second-line DST in settings where the need is greatest.
PMCID: PMC4167337  PMID: 25225802
13.  MiR-592 Regulates the Induction and Cell Death-Promoting Activity of p75NTR in Neuronal Ischemic Injury 
The Journal of Neuroscience  2014;34(9):3419-3428.
The neurotrophin receptor p75NTR has been implicated in mediating neuronal apoptosis after injury to the CNS. Despite its frequent induction in pathologic states, there is limited understanding of the mechanisms that regulate p75NTR expression after injury. Here, we show that after focal cerebral ischemia in vivo or oxygen–glucose deprivation in organotypic hippocampal slices or neurons, p75NTR is rapidly induced. A concomitant induction of proNGF, a ligand for p75NTR, is also observed. Induction of this ligand/receptor system is pathologically relevant, as a decrease in apoptosis, after oxygen–glucose deprivation, is observed in hippocampal neurons or slices after delivery of function-blocking antibodies to p75NTR or proNGF and in p75NTR and ngf haploinsufficient slices. Furthermore, a significant decrease in infarct volume was noted in p75NTR−/− mice compared with the wild type. We also investigated the regulatory mechanisms that lead to post-ischemic induction of p75NTR. We demonstrate that induction of p75NTR after ischemic injury is independent of transcription but requires active translation. Basal levels of p75NTR in neurons are maintained in part by the expression of microRNA miR-592, and an inverse correlation is seen between miR-592 and p75NTR levels in the adult brain. After cerebral ischemia, miR-592 levels fall, with a corresponding increase in p75NTR levels. Importantly, overexpression of miR-592 in neurons decreases the level of ischemic injury-induced p75NTR and attenuates activation of pro-apoptotic signaling and cell death. These results identify miR-592 as a key regulator of p75NTR expression and point to a potential therapeutic candidate to limit neuronal apoptosis after ischemic injury.
PMCID: PMC3935094  PMID: 24573298
ischemia; microRNA; neurotrophin; NGF; p75NTR; stroke
14.  A comparison of functional brain changes associated with surgical versus behavioral weight loss 
Obesity (Silver Spring, Md.)  2013;22(2):337-343.
Few studies have examined brain changes in response to effective weight loss; none have compared different methods of weight-loss intervention. We compared functional brain changes associated with a behavioral weight loss intervention to those associated with bariatric surgery.
15 obese participants were recruited prior to adjustable gastric banding surgery and 16 obese participants were recruited prior to a behavioral diet intervention. Groups were matched for demographics and amount of weight lost. fMRI scans (visual food motivation paradigm while hungry and following a meal) were conducted before, and 12 weeks after surgery/behavioral intervention.
When compared to bariatric patients in the pre-meal analyses, behavioral dieters showed increased activation to food images in right medial PFC and left precuneus following weight loss. When compared to behavioral dieters, bariatric patients showed increased activation in in bilateral temporal cortex following the weight loss.
Behavioral dieters showed increased responses to food cues in medial PFC – a region associated with valuation and processing of self-referent information – when compared to bariatric patients. Bariatric patients showed increased responses to food cues in brain regions associated with higher level perception—when compared to behavioral dieters. The method of weight loss determines unique changes in brain function.
PMCID: PMC3946492  PMID: 24115765
functional MRI; bariatric surgery; neuroscience; weight loss; neuroimaging
15.  Enhanced Fitness of Adult Spermatogonial Stem Cells Bearing a Paternal Age-Associated FGFR2 Mutation 
Stem Cell Reports  2014;3(2):219-226.
Pathogenic de novo mutations increase with fathers’ age and could be amplified through competition between genetically distinct subpopulations of spermatogonial stem cells (SSCs). Here, we tested the fitness of SSCs bearing wild-type human FGFR2 or an Apert syndrome mutant, FGFR2 (S252W), to provide experimental evidence for SSC competition. The S252W allele conferred enhanced FGFR2-mediated signaling, particularly at very low concentrations of ligand, and also subtle changes in gene expression. Mutant SSCs exhibited improved competitiveness in vitro and increased stem cell activity in vivo upon transplantation. The fitness advantage in vitro only occurred in low concentrations of fibroblast growth factor (FGF), was independent of FGF-driven proliferation, and was accompanied by increased response to glial cell line-derived neurotrophic factor (GDNF). Our studies provide experimental evidence of enhanced stem cell fitness in SSCs bearing a paternal age-associated mutation. Our model will be useful for interrogating other candidate mutations in the future to reveal mechanisms of disease risk.
Graphical Abstract
•FGFR2-mediated signaling regulates SSC self-renewal•Age-associated Apert syndrome FGFR2 mutation confers a fitness advantage to SSCs•Mutant FGFR2 enables SSCs to withstand limiting GDNF•Excessive growth factor exposure impairs SSC self-renewal signals
Seandel and colleagues address whether competition among stem cells in the testis mediates the increased frequency of mutations as the father ages. Using a paternal age-associated human FGFR2 mutation in Apert syndrome delivered to mouse spermatogonial stem cells as a model, their study demonstrates enhanced self-renewal activity of mutant cells versus wild-type populations. Furthermore, mutant cells exhibit higher sensitivity to limited growth factors, suggesting a selection mechanism mediated by the aged niche.
PMCID: PMC4176532  PMID: 25254335
16.  Electrophysiological and Hemodynamic Responses to Reward Prediction Violation 
Neuroreport  2009;20(13):1140-1143.
Anterior cingulate cortex has been functionally linked to the detection of outcomes that are worse than expected using both scalp electrophysiological (ERP) and hemodynamic (fMRI) responses. The current study used a reward prediction violation design acquired both ERP and fMRI data from the same participants in different sessions. Both the medial frontal negativity (MFN) ERP response and anterior cingulate cortex hemodynamic activity differentiated between reward delivery and expectation with the largest MFN and anterior cingulate cortex response when predicted rewards were not delivered. Inverse modeling placed the MFN source near the anterior cingulate cortex hemodynamic activation. The fMRI study also showed increased striatal response to rewards regardless of prediction indicating dissociation of neural processing of reward and reward expectation.
PMCID: PMC4095766  PMID: 19690501
Reward; Event Related Potentials; fMRI; anterior cingulate cortex
17.  Discovery of a Potent, Dual Serotonin and Norepinephrine Reuptake Inhibitor 
ACS Medicinal Chemistry Letters  2013;4(6):560-564.
The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug–drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions.
PMCID: PMC4027471  PMID: 24900709
SERT; NET; dual; reuptake inhibitor; SNRI; SERT RO; α-MMT; pain
18.  Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats 
PLoS ONE  2014;9(5):e98401.
We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Leprfa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Leprfa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Leprfa/fa rats that received probiotics than in rats fed the placebo. Zucker-Leprfa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Leprfa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean+/fa rats. Serum TNF-α levels decreased in the Zucker-Leprfa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Leprfa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats.
PMCID: PMC4031175  PMID: 24852284
19.  Trkb Signaling in Pericytes Is Required for Cardiac Microvessel Stabilization 
PLoS ONE  2014;9(1):e87406.
Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb−/−) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb−/− embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.
PMCID: PMC3909185  PMID: 24498100
20.  Nicotine Increases Cerebellar Activity during Finger Tapping 
PLoS ONE  2013;8(12):e84581.
Nicotine improves performance on several cognitive and sensorimotor tasks. The neuronal mechanisms associated with these changes in performance are, however, largely unknown. Functional magnetic resonance imaging (fMRI) was used to examine the effect of nicotine on neuronal response in nineteen healthy subjects while they performed an auditory-paced finger tapping task. Subjects performed the task, after receiving either a nicotine patch or placebo treatment, in a single blind, crossover design. Compared to placebo, nicotine treatment increased response in the cerebellar vermis. Increased vermal activity, in the absence of changes in other task-related regions suggests specificity in nicotine’s effects.
PMCID: PMC3866129  PMID: 24358367
21.  Safety and Immunomodulatory Effects of Three Probiotic Strains Isolated from the Feces of Breast-Fed Infants in Healthy Adults: SETOPROB Study 
PLoS ONE  2013;8(10):e78111.
We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers’ feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects.
Trial Registration NCT01479543
PMCID: PMC3810271  PMID: 24205115
22.  Nicotine Increases Brain Functional Network Efficiency 
NeuroImage  2012;63(1):73-80.
Despite the use of cholinergic therapies in Alzheimer’s disease and the development of cholinergic strategies for schizophrenia, relatively little is known about how the system modulates the connectivity and structure of large-scale brain networks. To better understand how nicotinic cholinergic systems alter these networks, this study examined the effects of nicotine on measures of whole-brain network communication efficiency. Resting-state fMRI was acquired from fifteen healthy subjects before and after the application of nicotine or placebo transdermal patches in a single blind, crossover design. Data, which were previously examined for default network activity, were analyzed with network topology techniques to measure changes in the communication efficiency of whole-brain networks. Nicotine significantly increased local efficiency, a parameter that estimates the network’s tolerance to local errors in communication. Nicotine also significantly enhanced the regional efficiency of limbic and paralimbic areas of the brain, areas which are especially altered in diseases such as Alzheimer’s disease and schizophrenia. These changes in network topology may be one mechanism by which cholinergic therapies improve brain function.
PMCID: PMC3429645  PMID: 22796985
nicotine; acetylcholine; graph theory; small-world; network; fMRI
23.  Comparative proteomic profiling of dystroglycan-associated proteins in wild type, mdx and Galgt2 transgenic mouse skeletal muscle 
Journal of proteome research  2012;11(9):4413-4424.
Dystroglycan is a major cell surface glycoprotein receptor for the extracellular matrix in skeletal muscle. Defects in dystroglycan glycosylation cause muscular dystrophy and alterations in dystroglycan glycosylation can impact extracellular matrix binding. Here we describe an immunoprecipitation technique that allows isolation of beta dystroglycan with members of the dystrophin-associated protein complex (DAPC) from detergent solubilized skeletal muscle.
Immunoprecipitation, coupled with shotgun proteomics, has allowed us to identify new dystroglycan-associated proteins and define changed associations that occur within the DAPC in dystrophic skeletal muscles. In addition, we describe changes that result from overexpression of Galgt2, a normally synaptic muscle glycosyltransferase that can modify alpha dystroglycan and inhibit the development of muscular dystrophy when it is overexpressed. These studies identify new dystroglycan-associated proteins that may participate in dystroglycan’s roles, both positive and negative, in muscular dystrophy.
PMCID: PMC3436944  PMID: 22775139
24.  Evaluation of the Brief Questionnaire of Smoking Urges in Black Light Smokers 
Nicotine & Tobacco Research  2012;14(9):1110-1114.
This study evaluated the factor structure of the Brief Questionnaire of Smoking Urges (QSU-Brief) within a sample of Black light smokers (1–10 cigarettes per day).
The QSU-Brief was administered to 540 (mean age = 46.5; 66.1% women) urban Black light smokers upon entering a smoking cessation clinical trial. An exploratory factor analysis (EFA) was conducted to evaluate the factor structure of this 10-item measure.
An EFA indicated that as in other samples, the construct of craving in a Black sample is defined by 2 factors; 1 factor emphasizing the positive reinforcement of smoking and the other factor emphasizing the negative reinforcement properties of smoking.
Findings largely replicate a 2-factor structure of craving seen in smokers from other racial/ethnic groups, demonstrating the clinical utility of the QSU-Brief in measuring craving in Black light smokers.
PMCID: PMC3432274  PMID: 22241828
25.  Differential Epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu, Nepal: A Matched Case Control Investigation in a Highly Endemic Enteric Fever Setting 
Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.
Methodology and principal findings
A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.
Conclusions and significance
Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.
Author Summary
Enteric fever, caused by ingestion of bacteria Salmonella Typhi or Salmonella Paratyphi A, is common in regions with poor water quality and sanitation. We sought to identify individual-level risks for infection in Kathmandu, Nepal, a region endemic for enteric fever. In this study, we enrolled patients presenting to hospital who were blood-culture positive for enteric fever and a series of community controls matched for age, gender and residential ward. Our findings suggest that while some risks for infection with S. Typhi and S. Paratyphi A overlap, these organisms also have distinctive routes of infection in this setting; poor water and socioeconomic status seemed more influential in infection with S. Typhi, whereas food consumption habits and migratory status were shown to play a larger role in infection with S. Paratyphi A. Additionally, serological evaluation of IgG levels against the Vi (Salmonella Typhi) and the O:2 (Salmonella Paratyphi A) antigens demonstrated high titers against both antigens throughout life, suggesting frequent and constant exposure to these organisms in Kathmandu. As major improvements in sanitation infrastructure are unlikely in this setting, we recommend water treatment and storage-based prevention strategies, as well as street food quality regulation, and the promotion of vaccination with existing typhoid vaccines.
PMCID: PMC3749961  PMID: 23991240

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